Cryopreservation of haptenized tumor cells

ABSTRACT

A method of preserving haptenized tumor cells is described. The method employs a freezing medium containing an effective amount of sucrose and human serum albumin in an isotonic buffered saline solution. Cryogenically preserving haptenized cells in such a medium has been found to maintain the integrity of the tumor cells during storage. The haptenized tumor cells also retain cell-associated antigens and haptens, and are as immunogenic, i.e., capable of inducing immunotherapeutic response, as fresh vaccine in a mouse model of metastatic disease. In a specific embodiment, haptenized cells are exposed to a solution of 8% sucrose, 10% human serum albumin in Hank&#39;s buffered solution, and then frozen to −80° C. overnight and then stored in a liquid nitrogen freezer. Methods of storing haptenized tumor cells and compositions are also provided.

FIELD OF THE INVENTION

The invention relates to compositions and methods for cryo-preservationof haptenized tumor cells. The tumor cell compositions are particularlysuitable for an immunotherapeutic vaccine.

BACKGROUND OF THE INVENTION

In blood transfusion, bone marrow transplantation, immuno-therapeuticvaccine preparation, or other cell preparations ex vivo, one of theprincipal problems encountered is that of the preservation of cells. Itis critical to be able to preserve cells, under good conditions ofviability, for time periods compatible with clinical production andstorage, and to make it possible to analyze cell preparations. The mostcommonly used method of long-term preservation of cells is to freeze andsubsequently thaw them. However, during the freezing of cells, lysis ofcells and loss of cell integrity may occur. This is often observed bythe decrease in intact tumor cells and the concomitant increase in theamount of non-intact tumor cells in a sample of tumor cells. Thisproblem can be even more complex when the cells have been modified oraltered prior to preservation, and when the cells are obtained byproteolytic digestion of a tissue or tumor specimen. Preservation ofcells under less extreme conditions, for example on ice (about 0° C.),refrigerated (about 4° C.), or at room temperature, prior to use, isalso difficult as these storage conditions are effective only for aperiod of hours.

Immunotherapy

The preservation of cells, especially their immunogenicity, is importantis in immunotherapy of cancer using tumor cells. The aim of theimmunotherapy is to evoke an immune response to the tumor, or tovaccinate against new tumors, by administering tumor cells or tumor cellextracts to the cancer patient. The tumor cells in the compositionshould contain antigens that are also present in the tumor to betreated, so that the immune response elicited against the antigens inthe composition is effective against the tumor. Generally, the cells arerecovered from tumors, suspended in a cryopreservation medium and frozenuntil used for the vaccine preparation. When needed, the cells arethawed, and then stored at temperatures ranging from about 0° C. (onice) to room temperature until administration.

Immunotherapy regimens using unmodified intact tumor cells prepared fromtumors taken from the patient, i.e., autologous tumor cells, have beenextensively described in the literature (see, e.g., Berd et al., CancerResearch 1986;46:2572-2577; Hoover et al., Cancer 1985;55:1236-1243; andU.S. Pat. No. 5,484,596 to Hanna et al.). Alternative vaccinecompositions based on disrupted cells have also been suggestedincluding, e.g., tumor membranes (see, e.g., Levin et al., In: HumanTumors in Short Term Culture: Techniques and Clinical Applications, P.P. Dendy, Ed., 1976, Academic Press, London, pp. 277-280) or tumorpeptides extracted from tumors (see, e.g., U.S. Pat. No. 5,550,214 toEberlein, and U.S. Pat. No. 5,487,556 to Elliot et al.). The tumor cellscan also be modified in some manner to alter or increase the immuneresponse (see, e.g., Hostetler et al., Cancer Research1989;49:1207-1213; and Muller et al., Anticancer Research 1991; 11:925-930).

Haptenized Tumor Cell Vaccines

One particular form of tumor cell modification that has a pronouncedeffect on immunotherapy is coupling of a hapten to the tumor cells. Anautologous whole-cell vaccine modified with the hapten dinitrophenyl(DNP) has been shown to produce inflammatory responses in metastaticsites of melanoma patients. Adjuvant therapy with DNP-modified vaccineproduces markedly higher post-surgical survival rates than thosereported after surgery alone. U.S. Pat. No. 5,290,551 to Berd disclosesand claims vaccine compositions comprising haptenized melanoma cells.Melanoma patients who were treated with these cells developed a strongimmune response. This response can be detected in a delayed-typehypersensitivity (DTH) response to haptenized and non-haptenized tumorcells. More importantly, the immune response resulted in increasedsurvival rates of melanoma patients.

Haptenized tumor cell vaccines have also been described for other typesof cancers, including lung cancer, breast cancer, colon cancer,pancreatic cancer, ovarian cancer, and leukemia (see InternationalPatent Publication Nos. WO 96/40173 and WO 00/09140, and U.S. Pat. No.6,333,028, and the associated techniques and treatment regimensoptimized (see International Patent Publication Nos. WO 00/38710, WO00/31542, WO 99/56773, WO 99/52546, and WO 98/14206). For example, ithas been shown that the addition of human serum albumin (HSA) increasesthe stability of haptenized tumor cell preparations (see WO 00/29554 andU.S. Pat. No. 6,248,585).

It has also been found that haptenization of tumor cell extracts such asplasma membranes and peptides can yield potent immunotherapy vaccines(see International Patent Publication Nos. WO 96/40173 and WO 99/40925,both by Berd et al.).

For haptenized vaccines, the search for storage conditions that preservethe stability of the haptenized cells or extracts also have to take intoaccount that some haptenization reactions may alter or affect the cellviability or integrity. Previous work has suggested that if no measuresare taken to increase the stability of haptenized melanoma vaccinepreparations, they might have a cell integrity duration of less thanfour hours after hapten modification. Also, some haptens orhaptenization procedures render the cells more fragile than others. Forexample, while preparations of DNP-modified cells can be stable for atleast 18 hours when stored at 4° C., some procedures for sulfanilic acid(SA) conjugation render the cells more fragile, and the SA-modifiedcells may in some cases only be stable for less than 2 hours at 4° C.

However, whether utilizing modified or unmodified tumor cells, in orderto elicit a successful immune response against the tumors of the patientafter administration, the amount and immunogenicity of the antigens inthe tumor cell composition should be retained as much as possible duringpreparation and storage of the composition. The tumor antigens shouldalso remain associated with the cells.

Thus, there is a need in the art for an effective treatment for cells tobe stored and preserved prior to delivery as an immunotherapy vaccine.There is also a need for a treatment that preserves the integrity,antigen-content and immunogenicity of such cells for vaccines prior toadministration, and methods for designing tumor cell preparations andformulations to obtain optimal immune response. The present inventionadvantageously addresses these and other needs in the art.

SUMMARY OF THE INVENTION

The present invention is based, in part, on a cryopreservation methodfound to preserve a haptenized tumor cell vaccine in terms of the numberof intact tumor cells; the density of various tumor-cell associatedantigens, including haptens; and the in vivo immunogenicity andimmunotherapeutic potential of the tumor cell vaccine. The presentinvention therefore advantageously provides a method of treating tumorcells or tumor cell extracts for their preservation and/or storage priorto use in anti-tumor vaccines.

Accordingly, the invention provides a method of preserving haptenizedtumor cells, which method comprises: (i) contacting the haptenized tumorcells with a freezing medium, wherein the freezing medium comprisessucrose, human serum albumin and an isotonic buffered solution; and (ii)freezing the tumor cells, whereby the immunogenicity of the tumor cellsis preserved. In one embodiment, the isotonic buffered saline solutionis Hank's buffered solution. For example, the freezing medium maycomprise an 8% sucrose, 10% human serum albumin-supplemented Hank'sbuffered solution. The storage temperature can be from about −20° C. toabout −196° C., preferably −80° C. to about −196° C. In one embodiment,at least 70%, preferably at least 90%, of the level of at least onetumor cell-associated antigen (TCAA) is preserved after about 3 monthsstorage at a temperature, e.g., of −80° C. or less. In anotherembodiment, at least 50%, preferably at least 70%, of the haptenizedtumor cells are preserved intact after about 3 months storage, e.g., ata temperature −80° C. or less. The tumor cells may, for example, bemelanoma cells, ovarian cancer cells, colorectal cancer cells, smallcell lung cancer cells, kidney cancer cells, breast cancer cells, orleukemia cells. In a particular embodiment, the tumor cells are melanomacells. The tumor cells are haptenized with at least one hapten, whichcan be selected from, e.g., DNP, TNP, and sulfanilic acid. In aparticular embodiment, the hapten is DNP. In another particularembodiment, the tumor cells are haptenized with at least two differenthaptens.

The invention also provides for a method of storage for haptenized tumorcells for use in a vaccine, which method comprises storing a haptenizedtumor cells and freezing medium composition at a temperature below thefreezing temperature for at least 3 months. In one embodiment, thetemperature is from about −80° C. to −196° C. The tumor cells may behaptenized with, for example, at least one hapten selected from DNA andsulfanilic acid.

The invention also provides for a composition comprising haptenizedtumor cells for use in a vaccine and freezing medium, wherein thefreezing medium comprises sucrose, human serum albumin and an isotonicbuffered saline solution. Preferably, the freezing medium comprises 8%sucrose, 10% human serum albumin and the isotonic buffered salinesolution is Hank's buffered solution. The tumor cells can be, forexample, melanoma cells, ovarian cancer cells, colorectal cancer cells,small cell lung cancer cells, kidney cancer cells, breast cancer cells,or leukemia cells. In a particular embodiment, the tumor cells aremelanoma cells. The tumor cells are haptenized with at least one hapten,which can be selected from, e.g., DNP, TNP, and sulfanilic acid. In aparticular embodiment, the hapten is DNP. In another particularembodiment, the tumor cells are haptenized with at least two differenthaptens.

The Drawings, Detailed Description, and Examples will further explainthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relapse-free survival rate of mice after surgicalremoval of primary 410.4 mammary adenocarcinoma tumors and treatmentwith cryogenically preserved irradiated DNP-modified tumor cells ascompared to control mice treated with saline.

FIG. 2 shows the relapse-free survival rate of mice treated withcryogenically preserved haptenized tumor cell vaccine as compared tomice treated with fresh haptenized tumor cell vaccine.

FIG. 3 shows the relapse-free survival rate of mice treated withcryogenically preserved haptenized tumor cell vaccine as compared tomice treated with cryogenically preserved non-haptenized tumor cellvaccine.

FIG. 4 shows the relapse-free survival rate of mice treated withcryogenically preserved haptenized irradiated tumor cell vaccine ascompared to mice treated with cryogenically preserved haptenizednon-irradiated vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention advantageously provides a new cryopreservationmethod that stabilizes haptenized tumor cells or tumor cell extracts forstorage and maintains immunogenicity, the level of cell-associatedantigens, and/or the integrity of the tumor cells. Provided are alsocompositions formed by such a method. Advantages of the present methodinclude that the method allows for storing tumor cells for months afterpreparation without risking substantial loss of intact cells,cell-associated antigens, and/or immunogenicity. For example, thecryopreservation method may not result in any significant loss of thedensity of various tumor cell associated antigens (TCAA). The preservedtumor cells are sufficiently immunogenic to provide protective immunity,and may retain the same immunogenicity, as assessed by an in vivo assay,as a fresh preparation of the same type of tumor cells. Thus, the methodof the invention provides an additional time window during which a tumorcell composition may be shipped to a clinic, examined for qualitycontrol, or subjected to further manipulation or analysis, prior topatient administration as a vaccine.

The method for the preservation and/or storage of tumor cells comprisescontacting the cells with a freezing medium comprising an effectiveamount of sucrose and human serum albumin, and then freezing the cells.The cells can be stored at any temperature below the freezingtemperature of the composition. Non-limiting examples of storagetemperatures include below −10° C., preferably below −20° C., even morepreferably below −80° C. Particularly preferred temperatures are in therange from about −80° C. to about −196° C. Suitable storage mediaincludes liquid nitrogen and freezers capable of maintaining suitabletemperatures. The cryopreservation method of the invention is suitablefor treatment of, e.g, any haptenized tumor cell such as, e.g.,haptenized tumor cells derived from melanoma, ovarian cancer, small celllung cancer, colon cancer, leukemia, or lymphoma, or haptenized membraneextracts of such tumor cells.

It has been found that preserved haptenized tumor cells that have beentreated with freezing medium and cryopreserved by the method of thepresent invention can be utilized in vaccines due to their retention ofcell characteristics and preservation of surface haptenization, such asDNP, and antigens such as MHC1, even if the cells do not exclude trypanblue. Preferably, in a solution of freezing medium treated cells thatare frozen to about −80° C. to −196° C., more than 50%, preferably atleast 60%, and even more preferably at least 70% of the tumor cellsremain intact, and more than 50% preferably more than 80%, and even morepreferably more than 90% of a tumor cell-associated antigen is preserved(present on the cells) after storage. Although the cells may be storedfor any suitable period of time, it is preferred that the tumor cells bestored for up to 3 months. In a specific embodiment, the tumor cells arehuman tumor cells.

The freezing medium of the present invention is a composition comprisingan effective amount of sucrose and human serum albumin and an isotonicbuffered saline solution to stabilize the haptenized tumor cells andpreserve the antigenicity of the haptenized tumor cells during thefreezing process. In a preferred embodiment, the freezing medium iscomposed of sucrose, human serum albumin and Hanks solution. The use ofsucrose and human serum albumin and an isotonic buffered saline solutionin the freezing medium preserves one or more of the antigenicity,immunogenicity and stability during cryopreservation while maintainingthe ability of the vaccine to be used in humans. By contrast, otherfreezing medium additives are not usable with haptenized tumor cellvaccines. For example, the use of dextran in the freezing medium in acryopreserved haptenized tumor cell vaccine resulted in a vaccine thatwas unusable in human as it induced anaphylaxis in mice.

In the present invention, the sucrose content preferably ranges fromabout 0.1% to about 40%. More preferably, the sucrose content rangesfrom about 1% to about 20%. Even more preferably, the sucrose contentranges from about 5% to about 15%. Most preferably, the sucrose contentis about 8%.

In addition to sucrose, the freezing medium can comprise human serumalbumin and an isotonic buffered saline solution. Preferably, the humanserum albumin content ranges from about 1% to about 30%. Morepreferably, the human serum albumin content ranges from about 5% toabout 15%. Even more preferably, the human serum albumin content isabout 10%. Hanks buffered solution or HBSS, a well-known buffersolution, is discussed in more detail below.

In a preferred embodiment, Hanks buffered solution is included as anisotonic buffered saline in the freezing medium. The skilled artisanwill understand that other buffered salt solutions may be utilized, forexample PBS. The formulations section, below, lists non-limitingexamples of buffered salt solutions. A preferred freezing medium of theinvention is a composition that comprises about 8% sucrose and about 10%human serum albumin in Hanks solution.

It will be appreciated by one skilled the art that the freezing processincludes the reduction of the temperature of the sample to the desiredtemperature, the maintenance of the desired temperature during storageof the sample, and thawing the sample for further use as a vaccine.Depending on the type of tumor cells, e.g., from different types ofcancers, and the intended use, there may be variations as to thetemperature reduction rate and other parameters. Based on the presentdisclosure, such parameters are easily recognized by a person skilled inthe art, who can optimize stability and preservation of antigenicity tosuit desired storage and sample conditions. In addition, the levels ofpurity and sterility for each intended use can be determined, and thepreparation and freezing process optimized accordingly. While thehaptenized tumor cells may be brought into contact with the freezingmedium by any suitable method, suspending the haptenized tumor cells inthe freezing medium is preferred.

The cryopreservation or freezing of haptenized tumor cells occurs whenthe haptenized tumor cells and freezing medium mixture is reduced intemperature relative to the temperature at which the freezing medium andhaptenized tumor cells were contacted. Preferably, the temperature ofthe mixture is reduced to about −20° C., and then to about −196° C., andmaintained at such temperature for the length of the desired storage.The longer the desired storage time, the lower the storage temperatureshould be utilized to improve yield. The haptenized tumor cells arepreferably stored in freezing medium at about −196° C., especially forlong term storage. For short and medium time periods, higher storagetemperatures can also be used. Preferably, temperatures above about −80°C. are not utilized for medium term and short term storage. Forshort-term storage, the storage temperature is preferably not above −10°C., even more preferably not above about −20° C.

The temperature can be maintained by any method known in the art.Non-limiting examples of freezing methods include electric freezers thatcan maintain temperatures from about −20° C. to about −180° C. Suchfreezers are commercially available. One such supplier is TermaForma ofMarietta, Ohio. In addition, freezers can maintain ultra-coldtemperatures by non-electric methods, which is also known in the art.For example, dry ice (frozen carbon dioxide at about −78° C.) can beused to maintain cold temperatures. Another example is liquid nitrogen(−196° C.) that is commonly used in ultra-cold freezers. Ultra-coldfreezers are commercially available, for example, from TermaForma ofMarietta, Ohio. In a preferred embodiment of the invention, the sampleis frozen overnight in a −80° C. freezer and then transferred to aliquid nitrogen freezer for storage. The storage time periods may extendfor many months, e.g., for up to 9 months, preferably up to 6 months,and even more preferably for up to 3 months. Storage for days or weeksis also encompassed in the method of the invention.

After cryopreservation, the cells may be used for preparing a tumor cellvaccine for administration to a patient in need thereof. Thepreservation method of the invention is particularly advantageous forsuch applications, since preserved cell can be maintained a longer timein storage without losing cell-associated antigens, immunogenicity orvaccine potency, thus permitting a longer period of time for qualityassurance (QA) and quality control (QC) of the vaccine beforeadministration to the patient. After thawing the cryopreserved vaccine,it may be used for various therapeutic applications in patients,including immunoprotection (treatment prior to tumor development, i.e.,“vaccination” against a tumor) and immunotherapy (treatment of a patientalready suffering from a tumor to prevent, e.g., tumor recurrence ormetastatic disease).

Haptenized tumor cells treated with the optimized concentration offreezing medium, comprising an effective amount of sucrose and humanserum albumin and then cryopreserved at about −20° C. to about −196° C.substantially retain their cell characteristics and substantiallypreserve surface haptenization and cell-associated antigens, asdetermined by flow cytometry. Preferably, the preservation offreezing-medium treated frozen cells is greater than the preservation ofthe same kind, number, and concentration of tumor cells contacted with acontrol medium for the same period of time and at the same temperatureand then frozen for the same amount of time and at the same temperature.

The various aspects of the invention will be set forth in greater detailin the following sections, directed to suitable medium and formulationsfor preserving haptenized tumor cells.

Definitions

The following defined terms are used throughout the presentspecification, and should be helpful in understanding the scope andpractice of the present invention.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value.

A “formulation” refers to an aqueous medium or solution for thepreservation of haptenized tumor cells, which is preferably directlyinjectable into an organism. An aqueous buffer will include salts orsugars, or both, at about an isotonic concentration. The formulation mayfurther comprise sucrose, as described herein.

“Human serum albumin” or “HSA” refers to a non-glycosylated monomericprotein consisting of 585 amino acid residues, with a molecular weightof 66 kD. Its globular structure is maintained by 17 disulphide bridges,which create a sequential series of 9 double loops (Brown, “Albuminstructure, function and uses”, Rosenoer, V. M. et al. (eds.), PergamonPress:Oxford, pp. 27-51, 1977). HSA may also be called human plasmaalbumin.

A “live” cell means a cell that has an intact cell, plasma, or “outer”membrane as assessed by Trypan Blue exclusion. A live cell may becapable of growth or maintenance, and division or multiplication, orattenuated, i.e., incapable of division and multiplication. A cell canbe rendered attenuated by, for example, irradiation.

“Dead” cells mean cells that do not exclude Trypan Blue as assessed in aTrypan Blue exclusion experiment, and that are incapable of division ormultiplication. A “dead” cell can be prepared by, e.g., freezing a livecell. A dead cell appears intact, e.g., by microscopic inspection,meaning that the cellular shape resembles that of a live cell. A “fixed”cell is one example of a dead cell.

A “lysed” cell is no longer intact, meaning that the cellular shape doesnot resemble that of a live cell.

A “preserved” cell is a cell that is not lysed. A preserved cell can belive or dead. The cell may or may not exclude Trypan Blue, but retainsits level of cell-associated antigens, preferably antigens present inthe cell membrane, or its immunogenicity over time better than a cellthat is not similarly preserved.

“Immunogenicity” means the ability of a tumor cell or tumor cell extractto evoke an immune response directed to the tumor cell or extract.Generally, immunogenicity is higher for a tumor cell in which theimmunogenic molecules are intact. Whether a haptenized tumor cellpreparation is immunogenic can be tested by, for instance, a DTH-assayor an in vivo assay in an experimental animal model. The animal modeldescribed in Example 5 demonstrates testing of human melanoma vaccines,and similar models can be applied for other tumor vaccines. Preservationof immunogenic molecules on tumor cells stored in accordance with theinvention can be determined by direct measurement of the immunogen, orindirectly by measuring preservation of other tumor associatedmolecules, which should correlate with preservation of the immunogen.

A “tumor cell associated antigen” (TCAA; also referred to as a tumorassociated antigen or “TAA”) is an antigen associated with a tumor cellin such a manner that an antibody or another component of a mammalianimmune system can recognize it. Preferably, although not necessarily,the TCAA is associated with the outer cell membrane or plasma membraneof a tumor cell. The TCAA is preferably, but not necessarily, tumorspecific in that the antigen is restricted to or over-expressed by atumor. Exemplary melanoma TCAAs include HLA Class I, CD45, GD3, S100,HMB45, and MART-1. In the context of the present invention, a TCAA of ahaptenized tumor cell vaccine can also be the cellular proteinsdecorated with hapten or haptens with which the tumor cells areassociated or conjugated. Such hapten TCAAs include, without limitation,DNP, TNP, and SA. The density or level of a TCAA or TCAAs on a tumorcell, a level that is also termed “antigenicity” herein, can be measuredusing, e.g., FACS analysis with antibodies directed against thetumor-associated antigens.

The term “cell recovery” or “cell recovery rate” is a measure of howmany cells are substantially intact, has a shape corresponding to orresembling that of a live cell, and/or has preserved antigenicity, aftera certain period of storage or incubation. When calculating cellrecovery, the number of cells at a certain time point or after a certainpreparation step is related to the number of cells at a reference timepoint or prior to the preparation step in question.

The phrase “pharmaceutically acceptable” refers to molecular entities,at particular concentrations, and compositions that are physiologicallytolerable and do not typically produce an allergic or similar untowardreaction, such as gastric upset, fever, dizziness and the like, whenadministered to a human or non-human animal. Preferably, as used herein,the term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government, or listed in the U.S.Pharmacopoeia or other generally recognized pharmacopoeia for use inhumans or non-human animals.

A “subject” is a human or a non-human animal who may receive haptenizedtumor cells formulated in a composition of the invention. Non-humananimals include domesticated pets, such as cats and dogs; farm animals,such as horses, cows, pigs, sheep, and goats; laboratory animals, suchas mice, rats, guinea pigs, and rabbits; etc.

An “anti-tumor response” is at least one of the following: tumornecrosis, tumor regression, tumor inflammation, tumor infiltration byactivated T lymphocytes, activation of tumor infiltrating lymphocytes,delayed-type hypersensitivity (DTH) response, or a clinical response.Clinical response criteria for anti-tumor response resulting fromtreatment according to the present invention include complete, partial,or mixed response, as well as stable disease. Other clinical responsesthat may be observed upon following the treatment of the invention isprolongation of time to relapse, or prolongation of survival.

A “formulation” refers to an aqueous medium or solution for thepreservation or administration, or both, of haptenized tumor cells ortumor cell extracts, which is preferably directly injectable into anorganism. The aqueous medium can include salts or sugars, or both, atabout an isotonic concentration.

A “vaccine composition” is a composition as set forth previously furthercomprising an adjuvant, including an immunostimulatory cytokine orlymphokine.

The terms “vaccine”, “immune therapy” and “immunotherapy” are usedherein interchangeably to administration of a composition comprising atumor cell preparation (preferably haptenized) to treat a cancer, e.g.,after surgical resection of the tumor.

“Efficacy of an immunotherapy” is the degree to which the immunotherapyelicits an anti-tumor response in an individual subject, or thepercentage of subjects in which an anti-tumor response develops as aresult of treatment. Preferably efficacy is determined by composition tocontrols that harbor the spontaneous tumor but receive either notherapy, sham therapy, or an alternative therapy.

A “tumor cell preparation” refers to isolated or purified tumor cells ora tumor cell extract for inclusion in a composition. “Hapten modified”means that the tumor cells (or extract) are chemically coupled(conjugated) to a hapten, as that term is understood immunology.

The term “treat” means to attempt to elicit an anti tumor responseagainst cells of the tumor, i.e., the cancer. An anti-tumor responseincludes, but is not limited to, increased time of survival, inhibitionof tumor metastasis, inhibition of tumor growth, tumor regression, anddevelopment of a delayed-type hypersensitivity (DTH) response tounmodified tumor cells.

As used herein, the term “control” generally describes a cell or cellsnot treated with freezing medium. The term control can also generallymean saline solution. More preferably, a control describes a compositionwhich in essentially all other aspects other than freezing mediumtreatment has been exposed to the same conditions, and is stored in thesame buffered medium and additional components.

Freezing Medium

As noted above, and demonstrated in the Examples, infra, it has beenunexpectedly discovered that exposure of tumor cells to an appropriatefreezing medium and then freezing the sample to the appropriatetemperature, e.g., below 0° C., preferably from −80 to −196° C.,maintains tumor cells, their antigenicity and their immunogenicityduring cryopreservation. This is especially advantageous for tumor cellsfor use in immunotherapy vaccine preparations. The freezing medium ofthe present invention is a composition comprising an effective amount ofsucrose and human serum albumin and an isotonic buffered saline solutionto stabilize the haptenized tumor cells and preserve the antigenicity ofthe haptenized tumor cells during the freezing process. In a preferredembodiment, the freezing medium is composed of sucrose, human serumalbumin and Hanks solution. Surprisingly, the use of sucrose and humanserum albumin and an isotonic buffered saline solution in the freezingmedium preserves the antigenicity, immunogenicity and stability duringcryopreservation while maintaining the ability of the vaccine to be usedin humans. Other components may be added to the freezing medium beyondsucrose, human serum albumin and Hank's solution, i.e., DMSO. However, apreferred embodiment of the freezing medium excludes DMSO.

In the present invention, the sucrose content may range from about 0.1%to about 40%. It is preferred that the sucrose content range from about1% to about 20%. It is more preferred that the sucrose content rangefrom about 5% to about 15%. A exemplified, preferred sucrose content is8%. In addition to sucrose the freezing medium requires human serumalbumin and an isotonic buffered saline solution. It is preferred thatthe human serum albumin content ranges from about 30% to about 1%. It ismore preferred that the human serum albumin content range from about 5%to about 15%. It is still more preferred that the human serum albumincontent is about 10%. Hanks buffered solution is a standard buffersolution and is discussed in more detail below. A preferred embodimentof the invention utilizes Hanks buffered solution in the freezingmedium. It is contemplated that a skilled artisan will understand thatother buffered salt solutions may be utilized, for example PBS. A verypreferred embodiment of the freezing medium is a composition that is 8%sucrose, 10% human serum albumin in Hanks solution.

Depending on the specific tumor cells to be stored, and theirmodification, if any, one of ordinary skill in the art can optimize thefreezing medium of the invention to their specific requirements. Such afreezing medium can be one that yields an increase in cell preservationrelative to a control for stored tumor cells. For example, such afreezing medium can be one that retains the amount of antigen-expressionand immunogenicity cells relative to a control. Preferably, the increasein preservation of the cells is statistically significant. In a verypreferred embodiment, the cells are then stored at about −196° C.conditions. In one embodiment, the cells are first stored in a −80° C.freezer and then transferred to liquid nitrogen. By this method thepreservation of antigen-expression and immunogenicity can besubstantially preserved in haptenized tumor cell vaccine. Preferably,the preservation of a tumor cell subjected to freezing medium treatmentand then frozen at −196° C. is greater than the same kind of tumor cellsstored in control medium for the same period of time, at the sametemperature.

The concentration of cells to be used during the freezing mediumtreatment step can be determined experimentally depending on the type ofcells or cell preparation used. However, a generally suitableconcentration is between 10⁵-10⁸ cells, more preferably between 10⁶ to10⁷ cells, and most preferably about 5×10⁶ cells, per millilitersolution. The solution is advantageously, although not necessarily,isotonic.

Tumor cell extracts such as membranes, tumor cell lysates where cellnuclei are removed, or simply lysed or disrupted cells, can be preservedaccording to similar procedures as described for intact, orsubstantially intact, tumor cells. Tumor cells or tumor cell extractsprocessed for use in an immunotherapy regimen as described below may besubjected to freezing medium treatment at any time during processing orformulation and cryopreserved. The concentration of cell membranes,lysed cells, or disrupted cells, is usually expressed as “cellequivalents”, or “c.e.”, herein.

For vaccines comprising haptenized tumor cells, freezing mediumtreatment and cryopreservation is preferably, although not necessarily,conducted after haptenization.

Tumor Cells

The tumor cells used in the present invention are prepared from tumorcells, e.g., obtained from tumors, or tissue or body fluids containingtumor cells, surgically resected or retrieved in the course of atreatment for a cancer. The sucrose freezing medium treatedcryopreserved tumor cells are useful in the preparation of, e.g., tumorcell vaccines for treating cancer, including metastatic and primarycancers. If used in a tumor cell vaccine, the preserved tumor cellsshould be incapable of growing and dividing after administration intothe subject, such that they are dead or substantially in a state of nogrowth. It is to be understood that “dead cells” means a cell which donot have an intact cell or plasma membrane and that will not divide invivo; and that “cells in a state of no growth” means live cells thatwill not divide in vivo. Conventional methods of suspending cells in astate of no growth are known to skilled artisans and may be useful inthe present invention. For example, cells may be irradiated prior to usesuch that they do not multiply. Tumor cells may be irradiated to receivea dose of 2500 cGy to prevent the cells from multiplying afteradministration. Alternatively, ethanol treatment may be used to createdead cells.

The tumor cells can be prepared from virtually any type of tumor. Thepresent invention contemplates the use of tumor cells from solid tumors,including carcinomas; and non solid tumors, including hematologicmalignancies. Examples of solid tumors from which tumor cells can bederived include sarcomas and carcinomas such as, but not limited to:fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblstoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Hematologic malignancies include leukemias, lymphomas,and multiple myelomas. The following are non limiting preferred examplesof tumor cells to be preserved according to the present invention:melanoma, including stage-4 melanoma; ovarian, including advancedovarian; small cell lung cancer; leukemia, including and not limited toacute myelogenous leukemia; colon, including colon metastasized toliver; rectal, colorectal, breast, lung, kidney, and prostate cancercells.

Tumor cell vaccines can be prepared from any of the tumor cell typeslisted above. Such tumor cell vaccines can comprise preserved cells,i.e., cells treated with ethanol according to the method of theinvention. Preferably, the vaccine comprises the same type of cells asthe tumor to be treated. Most preferably, the tumor cells areautologous, derived from the patient for whom treatment with the vaccineis intended. Vaccines comprising tumor cells prepared using the methodof the invention can used for treatment of both solid and non-solidtumors, as exemplified above. Thus, the invention includes “preserved”vaccines prepared from, and intended for treatment of, solid tumors,including carcinomas; and non solid tumors, including hematologicmalignancies. Preferred tumor types for vaccines include melanoma,ovarian cancer, colon cancer, and small cell lung cancer.

The tumor cells are preferably of the same type as, most preferablysyngeneic (e.g., autologous or tissue-type matched) to, the cancer thatis to be treated. For purposes of the present invention, syngeneicrefers to tumor cells that are closely enough related genetically thatthe immune system of the intended recipient will recognize the cells as“self”, e.g., the cells express the same or almost the same complementof HLA molecules. Another term for this is “tissue-type matched.” Forexample, genetic identity may be determined with respect to antigens orimmunological reactions, and any other methods known in the art.Preferably the cells originate from the type of cancer that is to betreated, and more preferably, from the same patient who is to betreated. The tumor cells can be, although not limited to, autologouscells dissociated from biopsy or surgical resection specimens, or fromtissue culture of such cells. Nonetheless, allogeneic cells and stemcells are also within the scope of the present invention.

Tumor cells for use in the present invention may be prepared as follows.Tumors are processed as described by Berd et al. (Cancer Res. 1986;46:2572; see also U.S. Pat. No. 5,290,551; U.S. patent application Ser.Nos. 08/203,004, 08/475,016, and 08/899,905). The cells are extracted bydissociation, such as by enzymatic dissociation with collagenase andDNase, or by mechanical dissociation such as with a blender, teasingwith tweezers, mortar and pestle, cutting into small pieces using ascalpel blade, and the like. Mechanically dissociated cells can befurther treated with enzymes as set forth above to prepare a single cellsuspension.

Tumor cells may also be prepared according to Hanna et al., U.S. Pat.No. 5,484,596. Briefly, tumor tissue is obtained from patients sufferingfrom the particular solid cancer from which the vaccine is to beprepared. The tumor tissue is surgically removed from the patient,separated from any non tumor tissue, and cut into small pieces, e.g.,fragments 2-3 mm in diameter. The tumor fragments are then digested tofree individual tumor cells by incubation in an enzyme solution. Afterdigestion, the cells are pooled and counted, and cell viability isassessed. If desired, a Trypan Blue exclusion test can be used to assesscell viability.

In addition, tumor cells can be prepared according to the followingprocedure (see Hanna et al., U.S. Pat. No. 5,484,596). The tissuedissociation procedure of Peters et al. (Cancer Research 1979; 39:13531360) can be employed using sterile techniques throughout under alaminar flow hood. Tumor tissue can be rinsed three times in thecentrifuge tube with HBSS and gentamicin and transferred to a petri dishon ice. Scalpel dissection removed extraneous tissue and the tumor areminced into pieces approximately 2 to 3 mm in diameter. Tissue fragmentsare placed in a 75 ml flask with 20-40 ml of 0.14% (200 units/ml)Collagenase Type 1 (Sigma C-0130) and 0.1% (500 Kunitz units/ml)deoxyribonuclease type 1 (Sigma D-0876) (DNAase 1, Sigma D-0876)prewarmed to 37° C. Flasks are placed in a 37° C. water bath withsubmersible magnetic stirrers at a speed that cause tumbling, but notfoaming. After a 30-minute incubation, free cells are decanted throughthree layers of sterile medium wet nylon mesh (166t: Martin Supply Co.,Baltimore, Md.) into a 50 ml centrifuge tube. The cells are centrifugedat 1200 rpm (250×g) in a refrigerated centrifuge for 10 minutes. Thesupernatant is poured off and the cell are resuspended in 5-10 ml ofDNAse (0.1% in HBSS) and held at 37° C. for 5-10 minutes. The tube isfilled with HBSS, washed by centrifugation, resuspended to 15 ml in HBSSand held on ice. The procedure is repeated until sufficient cells areobtained, usually three times for tumor cells. Cells from the differentdigests are then pooled, counted. Optionally, although not necessarily,cell viability is assessed by the Trypan Blue exclusion test.

The concentration of dissociated tumor cells can be adjusted to about5-10×10⁷/ml, or to about 5×10⁷ or 10×10⁷ cells per ml, in sucrosefreezing medium.

Tumor Cell Extracts

The tumor cells retrieved from tumors as described above may be furtherprocessed to yield tumor cell extracts for use in tumor cell vaccines.

To prepare tumor cell membranes for use in a vaccine, the followingprocedure can be used. Tumor cells are washed twice in Hanks balancedsalt solution. Cells are suspended in about five volumes of about 30 mMsodium bicarbonate buffer with about 1 mM phenyl methyl sulfonylfluoride and disrupted with a glass homogenizer. Residual intact cellsand nuclei are removed by centrifugation at about 1000×g. The membranesare pelleted by centrifugation at 100,000 g for 90 minutes. Themembranes are re-suspended in about 8% sucrose and frozen until needed,preferably, although not necessarily, at about −196° C. Other proceduresfor preparing tumor cell membranes are well known in the art asdescribed in, e.g., WO 96/40173 and WO 99/40925, both by Berd et al.These publications also describe the extraction of tumor cell peptides,which also can be used in immunotherapy vaccines.

Alternatively, whole tumor cell extracts can be prepared simply bylysing cells using any of the methods known in the art, for example,homogenization or suspending the cells in a detergent or hypotonicsolution, or cell lysis solution (e.g., Cytobuster™ from Novagen),containing additional components such as EDTA, protease inhibitors, andbuffering components, as desired.

Haptens

In one embodiment, the tumor cells or tumor cell extracts arehaptenized. For purposes of the present invention, virtually any smallprotein or other small molecule that fails to induce an immune responsewhen administered alone, may function as a hapten. A variety of haptensof quite different chemical structure have been shown to induce similartypes of immune responses, e.g., TNP (Kempkes et al., J. Immunol., 1991;147:2467); phosphorylcholine (Jang et al., Eur. J. Immunol., 1991;21:1303); nickel (Pistoor et al., J. Invest. Dermatol., 1995; 105:92);and arsenate (Nalefski and Rao, J. Immunol., 150:3806, 1993).Conjugation of a hapten to a cell to elicit an immune response maypreferably be accomplished by conjugation via ε-amino groups of lysineor —COOH groups. This group of haptens include a number of chemicallydiverse compounds: dinitrophenyl, trinitrophenyl,N-iodoacetyl-N′-(5-sulfonic 1-naphthyl) ethylene diamine,trinitrobenzene-sulfonic acid, dinitrobenzene sulfonic acid, fluoresceinisothiocyanate, arsenic acid benzene isothiocyanate, anddinitrobenzene-S-mustard (Nahas and Leskowitz, Cellular Immunol., 1980;54:241). Once armed with the present disclosure, skilled artisans wouldbe able to choose haptens for use in the present invention.

Haptenization

A variety of haptens of different chemical structure have been shown toinduce similar types of immune responses: e.g., dinitrophenyl (DNP);trinitrophenyl (TNP) (Kempkes et al., J. Immunol., 1991; 147:2467);phosphorylcholine (Jang et al., Eur. J. Immunol., 1991; 21:1303); nickel(Pistoor et al., J. Invest. Dermatol., 1995; 105:92); and arsenate(Nalefski and Rao, J. Immunol., 1993; 150:3806). Conjugation of a haptento a cell can, for example, be accomplished by conjugation via {tildeover (ε)}-amino groups of lysine or —COOH groups. This group of haptensinclude a number of chemically diverse compounds: halonitrobenzenes(including dinitrofluorobenzene, difluorodinitrobenzene,trinitrofluoro-benzene), N iodoacetyl N′ (5 sulfonic 1 naphthyl)ethylene diamine, nitrobenzene sulfonic acids (includingtrinitrobenzenesulfonic acid and dinitrobenzene sulfonic acid),fluorescein isothiocyanate, arsenic acid benzene isothiocyanate, anddinitrobenzene S mustard (Nahas and Leskowitz, Cellular Immunol., 1980;54:241).

In general, haptens include a “recognition group”, which is the groupthat interacts with an antibody. The recognition group is irreversiblyassociated with the hapten reactive group. Thus, when the haptenreactive group is conjugated to a functional group on the targetmolecule, the hapten recognition group is available for binding withantibody. Examples of different hapten recognition groups includewithout limitation to dinitiophenyl, trinitrophenyl, fluorescein, otheraromatics, phosphorylcholine, peptides, advanced glycosylationendproducts (AGE), carbohydrates, etc.

Haptens also include a functional group for conjugation to a substituenton an amino acid side chain of a protein or polypeptide. Amino acid sidechain groups that can be conjugated to hapten include, e.g., freecarboxylic acid groups in the aspartic acid or glutamic acid; the εamino group of lysine; the thiol moiety of cysteine; the hydroxyl groupof serine or tyrosine; the imidazole moiety of histidine; or the arylgroups of tryptophan, tyrosine, or phenylalanine. Hapten functionalgroups capable of reacting with specific amino acid side chains aredescribed below.

Functional groups reactive with primary amines. Hapten reactive groupsthat would form a covalent bond with primary amines present on aminoacid side chains would include, but not be limited to, acid chlorides,anhydrides, reactive esters, α,β-unsaturated ketones, imidoesters, andhalonitrobenzenes. Various reactive esters with the capability ofreacting with nucleophilic groups such as primary amines are availablecommercially, e.g., from Pierce (Rockford, Ill.).

Functional groups reactive with carboxylic acids. Carboxylic acids inthe presence of carbodiimides, such as EDC, can be activated, allowingfor interaction with various nucleophiles, including primary andsecondary amines. Alkylation of carboxylic acids to form stable esterscan be achieved by interaction with sulfur or nitrogen mustards, orhaptens containing either an alkyl or aryl aziridine moiety.

Functional groups reactive with aromatic groups. Interaction of thearomatic moieties associated with certain amino acids can beaccomplished by photoactivation of aryl diazonium compound in thepresence of the protein or peptide. Thus, modification of the aryl sidechains of histidine, tryptophan, tyrosine, and phenylalanine,particularly histidine and tryptophan, can be achieved by the use ofsuch a reactive functionality.

Functional groups reactive with sulfhydryl groups. There are severalreactive groups that can be coupled to sulfhydryl groups present on theside chains of amino acids. Haptens containing an α,β unsaturated ketoneor ester moiety, such as maleimide, provide a reactive functionalitythat can interact with sulfhydryl as well as amino groups. In addition,a reactive disulfide group, such as 2-pyridyldithio group or a5,5′-dithio-bis-(2-nitrobenzoic acid) group is also applicable. Someexamples of reagents containing reactive disulfide bonds includeN-succinimidyl 3-(2-pyridyl-dithio) propionate (Carlsson, et al.,Biochem J., 1978; 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzyl-thiosulfate, and 4succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)-toluene. Someexamples of reagents comprising reactive groups having a double bondthat reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidylm-maleimidobenzoate.

Other functional molecules include succinimidyl3-(maleimido)-propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfo-succinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-N-hydroxy-succinimide ester. Many of theabove-mentioned reagents and their sulfonate salts are available fromPierce (Rockford, Ill.).

Any hapten or combination of different haptens can be used in thecompositions of the invention. For example, in one embodiment, the samehapten recognition group is coupled to different amino acids throughdifferent functional groups. For example, the reagents dinitrobenzenesulfonic acid, dinitro phenyldiazonium, and dinitrobenzene S mustard,all form the dinitrophenyl hapten coupled to amino groups, aromaticgroups, and carboxylic acid groups, respectively. Similarly, an arsonicacid hapten can be coupled by reacting arsonic acid benzeneisothiocyanate to amino groups or azobenzenearsonate to aromatic groups.In another embodiment, the tumor cells are dual-haptenized, i.e., thesame tumor cell preparation is conjugated with two different haptens.The haptens may comprise reactive groups that react with differentfunctional groups on the tumor cell, such as different amino acids. Suchdual-haptenization is described in WO 00/38710 by Berd et al.

In yet another embodiment, the tumor cell can be bi-haptenized, i.e.,two or more aliquots of a single tumor cell preparation is each coupledto a different hapten and mixed prior to administration or administeredin conjunction with each other. Since, e.g., DNP modifies hydrophilicresidues of MHC-bound peptides (mainly lysine εamino groups) (Nahas andLesowitz, Cellular Immunol, 1980; 54:241), the second hapten couldadvantageously be conjugated to hydrophobic residues (such as tyrosineand histidine). Such haptens, binding proteins through an azo linkage,include sulfanilic acid, arsanilic acid, and phosphorylcholine. Otherhaptens such as and not limited to trinitrophenyl,N-iodoacetyl-N′-(5-sulfonic 1-naphthyl) ethylene diamine,trinitrobenzene-sulfonic acid, fluorescein isothiocyanate, arsenic acidbenzene isothiocyanate, trinitrobenzenesulfonic acid, sulfanilic acid,arsanilic acid, dinitrobenzene-S-mustard and combinations thereof may besimilarly used.

Modification of the prepared cells with a hapten may be performed byknown methods, e.g. by the method of Miller and Clanian (J. Immunol.1976; 117:151). The described procedure involves a 30 minute incubationof tumor cells with DNFB under sterile conditions, followed by washingwith sterile saline or Hanks/HSA. Other procedures for haptenization areknown in the art (see, e.g., International Patent Publications WO96/40173, WO 00/09140, WO 00/31542, WO 99/56773, WO 99/52546, WO99/40925, WO 98/14206, WO 00/295, all by Berd et al., and U.S. Pat. No.5,290,551 to Berd, hereby incorporated by reference in its entirety).The following procedures illustrate exemplary haptenization procedures.

DNP modification. Modification of the prepared cells with DNP or anotherhapten may be performed by known methods, e.g., by the method of Millerand Clanian (J. Immunol., 1976; 117:151), incorporated herein byreference in its entirety, which involves a 30 minute incubation oftumor cells with DNFB under sterile conditions, followed by washing withsterile saline or HBSS/HSA. For example, about 100 mg of DNFB (SigmaChemical Co., St. Louis, Mo.) can be dissolved in about 0.5 ml of 70%ethanol. About 99.5 ml of PBS is added. The solution is stirredovernight in a 37° C. water bath. The shelf life of the solution isabout 4 weeks. The cells are thawed and the pellet resuspended in 5×10⁶cells/ml in Hanks balanced salt solution. About 0.1 ml DNFB solution isadded to each ml of cells and incubated for about 30 minutes at roomtemperature.

SA modification. Modification of the prepared cells with SA may beperformed by known methods. For example, in one embodiment, sulfanilicacid (SA) is sterile filtered (0:2 μm), 10% sodium nitrite solution isadded, dropwise, to a SA solution of 100 mg of anhydrous SA dissolved in10 ml of 0.1 N HCl until saturation. (The saturation point correspondsapproximately to a final concentration of a sulfanilic acid diazoniumsalt of about 40 mM). The SA diazonium salt solution is then sterilefiltered (0.2 μm membrane), and diluted 1:8 (v/v) in HBSS (without HSA).If needed, the pH is adjusted to 7.2 by dropwise addition of 1N NaOH.The SA diazonium salt/HBSS solution is then sterilized by filtration(0.2 μm membrane). Pelleted tumor cells are resuspended in diazoniumsalt/HBSS solution to a final concentration of 5×10⁶ cells. The cellmixture is incubated for 5 minutes at room temperature. After the 5minute incubation period, the hapenization reaction is stopped by theaddition of 0.5 ml of a 25% HSA/HBSS solution to the cell mixture.

Formulations

The tumor cells and tumor cell extracts treated with freezing medium andcryopreserved according to the invention may be included in variousformulations. For example, sucrose freezing medium treated,cryopreserved tumor cells may, in haptenized form, be useful forpreparing tumor vaccines. The different components of such a formulationmay be mixed together, and then added to tumor cells. It is alsopossible to mix one or several of the components with the tumor cellsand then to add the remaining component(s). The preparation of theformulation and its addition of the tumor cells are preferably performedunder sterile conditions. Preferably, the tumor cells are subjected tofreezing medium treatment and cryopreservation before the finalformulation. However, one or more components to be included in the finalformulation may also be present before or during the sucrose freezingmedium treatment and cryopreservation step.

Persons skilled in the art may adapt the respective proportions of thecomponents of the medium according to the invention. As illustratedbelow, the proportions may be modified although certain concentrationranges are preferred.

Generally, an appropriate buffered medium is used for tumor cellformulation. In its essence, a buffered medium is an isotonic bufferedaqueous solution, such as phosphate buffered saline (PBS), Tris-bufferedsaline, or HEPES buffered saline. In a preferred embodiment, the mediumis a buffered cell culture medium such as plain Hank's medium (notcontaining phenol red), e.g., as solid commercially by Sigma ChemicalCo. (St. Louis, Mo., USA). Other tissue culture medium can also be used,including basal medium Eagle (with either Earle's or Hank's salts),Dulbecco's modified, Eagle's medium (DMEM), Iscove's modified Dulbecco'smedium (IMDM), Medium 199, Minimal Essential Medium (MEM) Eagle (withEarle's or Hank's salts), RPMI, Dulbecco's phosphate buffered salts,Earle's balanced salts (EBSS), and Hank's Balanced Salts (HBSS). Thesemediums can be supplemented, e.g., with glucose, Ham's nutrients, orHEPES. Other components, such as sodium bicarbonate and L-glutamine, canbe specifically included or omitted. Medium, salts, and other reagentscan be purchased from numerous sources, including Sigma, Gibco, BRL,Mediatech, and other companies.

Generally, human serum albumin (HSA) is also included, as describedbelow. In addition, a composition or formulation of the invention maycontain components in addition to HSA to further stabilize thehaptenized tumor cells. Examples of such components include, but are notlimited to, carbohydrates and sugars such as sucrose, glucose, and thelike, e.g., at a 5% concentration; medium to long chain polyols such asglycerol, polyethylene glycol, and the like, e.g., at 10% concentration;other proteins; amino acids; nucleic acids; chelators; proteolysisinhibitors; preservatives; and other components. Preferably, any suchconstituent of a composition of the invention is pharmaceuticallyacceptable.

Human Serum Albumin

In a preferred embodiment, the tumor cell formulations of the inventioncomprise a concentration or amount of a protein such as, e.g., albumin,which is effective to stabilize the tumor cells. An amount of proteineffective to stabilize the tumor cells may be added before and/or aftercryopreservation, or, in the case of haptenized tumor cells, beforeand/or after haptenization. In a preferred embodiment, the albumin ishuman serum albumin or HSA. HSA has been shown to stabilize solutions ofproteins, including protein antigens, and small organic molecules suchas hemin (Paige, A. G. et al., Pharmaceutical Res., 12:1883-1888, 1995;Chang, A. C. and R. K. Gupta, J., Pharm. Sci., 85:129-132, 1996;Niemeijer, N. R. et al., Ann. Allergy Asthma Immunol., 76:535-540, 1996;and Cannon, J. B. et al., PDA:J. Pharm. Sci. & Tech., 49:77-82, 1995),as well as haptenized tumor cell compositions (see WO 00/29554,corresponding to U.S. Pat. No. 6,248,585).

The HSA used within the framework of the present invention may be eitherof natural origin (purified HSA) or of recombinant origin (rHSA).Naturally, for delivery of a formulation in vivo, it is preferable touse an autologous or non-immunogenic serum albumin. Thus, for humantherapy, HSA is desirable and preferred. However, the skilled person canimmediately appreciate that any serum albumin can be used in thepractice of this invention, and, more particularly, any autologous serumalbumin can be used in connection with tumor cell vaccine for cancertreatment in any non-human animal as well. In a specific embodiment, aHuman Serum Albumin Solution (American Red Cross), which is a 25% HSAsolution, is used.

Advantageously, a recombinant or natural HSA is used which meets certainquality criteria (e.g., homogenetic, purity, stability). Thus, thepharmacopoeias set a number of parameters for the albumin solutions,namely a pH value, a protein content, a polymer and aggregate content,an alkaline phosphatase content, and a certain protein composition. Itimposes, furthermore, a certain absorbance, the compliance with testsfor sterility, pyrogens, and toxicity (see “Albumini humai solutio”,European Pharmacocpoeia (1984), 255). The use of an albumin compositioncorresponding to these criteria, although not essential, is particularlypreferred.

Generally, the HSA formulation of the invention is made by adding HSApowder or solution to the selected culture medium/balanced saltsolution, to achieve the desired final concentration, as set forthabove.

Additional information about the use of albumin in formulations of tumorcells, especially haptenized tumor cells, can be found in WO 00/29554,corresponding to U.S. Pat. No. 6,248,585.

Vaccine Preparation and Administration

The compositions of the invention may be administered in a mixture witha pharmaceutically acceptable carrier, selected with regard to theintended route of administration and standard pharmaceutical practice.Dosages may be set with regard to weight and clinical condition of thepatient. The proportional ratio of active ingredient to carriernaturally depends on the chemical nature, solubility, and stability ofthe compositions, as well as the dosage contemplated. The amounts to beused of the tumor cells of the invention depend on such factors as theaffinity of the compound for cancerous cells, the amount of cancerouscells present and the solubility of the composition. Any suitable route,including inoculation and injection modes using, for example,intradermal, intravenous, intraperitoneal, intramuscular, andsubcutaneous routes, may administer the compounds of the presentinvention. For example, the composition may be administered byintradermal injection into 3 contiguous sites per administration on theupper arms or legs, excluding limbs ipsilateral to a lymph nodedissection. In addition, the vaccine may be given by subcutaneousinjection close to the site of a tumor excision.

Tumor Cell Dose

Traditionally, the amount of tumor cells to be included in tumor cellvaccines has been determined based upon the number of viable, i.e.,Trypan Blue-excluding, cells (see, e.g., Hoover et al., 1985;55:1236-1243; and U.S. Pat. No. 5,484,596 to Hanna et al.). According toa preferred embodiment of the invention, the number of tumor cells inthe tumor cell vaccine to be administered to the patient is not basedupon the number of viable tumor cells, but on the total number of tumorcells, i.e., both live and “dead” cells as assessed by Trypan Blueexclusion.

The total cell counting procedure can be carried out by any suitablemethod known in the art. For example, cells can be counted manuallyusing a microscope and standard cell counting chambers, or by usingautomatic cell counters such as, e.g., Beckman Coulter cell counters.Since the method does not require distinguishing between live and “dead”cells, Trypan Blue and other means, which are selective for live or deadcells, can be omitted. The concentration of cells can then be adjustedby diluting the cells with a sterile solution so that a certain volumecorresponds to the number of cells to be injected into the patient, andthis volume aliquoted into storage vials.

In a preferred embodiment of the invention, the composition comprises avaccine comprising about 1×10⁴ to 1×10⁸, more preferably 1×10⁶ to about25×10⁶, even more preferably about 2.5×10⁶ to about 7.5×10⁶, tumor cellsor tumor cell equivalents suspended in a pharmaceutically acceptablecarrier or diluent, such as, but not limited to, Hank's solution (HBSS),saline, phosphate buffered saline, and water.

Adjuvants

In preferred embodiment, a tumor cell composition may be administeredwith an immunological adjuvant. While the commercial availability ofpharmaceutically acceptable adjuvants is limited, representativeexamples of adjuvants include Bacille Calmette-Guerin, BCG, or thesynthetic adjuvant, QS-21 comprising a homogeneous saponin purified fromthe bark of Quillaja saponaria, Corynebacterium parvum, (McCune et al.,Cancer 1979; 43:1619), and IL-12.

It will be understood that the adjuvant is subject to optimization. Inother words, the skilled artisan can engage in no more than routineexperimentation and determine the best adjuvant to use.

Immunostimulants and Combination Therapies

The tumor cell compositions may be co-administered with other compoundsincluding but not limited to cytokines such as interleukin-2,interleukin-4, gamma interferon, interleukin-12, GM-CSF. The tumor cellsand extracts of the invention may also be used in conjunction with othercancer treatments including but not limited to chemotherapy, radiation,antibodies, antisense oligonucleotides, and gene therapy.

EXAMPLES

The following examples are illustrative of the invention, but notlimiting thereof.

Example 1 Frozen Vaccine Processing

This Example describes the processing and storage of tumor cells. Thetumor cells are prepared from patient tumors, haptenized, and frozen.

Materials and Equipment

Collagenase (Sigma cat.#C1639 or C9722); Hanks Balanced Salt Solutionwithout phenol red (Gibco/BRL cat. #14175-095 or equivalent); EDTAdisodium (IBI cat.#IB70182, Sigma #E8008 or equivalent); sucrosecertified A.C.S. (Fisher Cat.#S5 3 or equivalent); PBS (calcium,magnesium free) (Sigma Cat.# D8537 or equivalent); DNFB (Sigma Cat.#D1529); Nalgene filter units (PES) 0.20 μm (Cat.#165-0020) orequivalent; Falcon 50 ml and 15 ml centrifuge tubes or equivlent; TrypanBlue 0.4% (Gibco/BRL Cat.#15-250-061 or equivalent); isopropanol;cryovials, 1.0 mL, externally threaded (VWR Cat. #66021-994, or FisherCat # 12-565-164N); #50 Mesh Screen; human albumin (HSA) 25%; Class IIBiological Safety Cabinet; Nalgene Cryo Container Cat. #5100-0001;hemacytometer; microscope.

Procedure

Although tumor cells from any solid tumor can be prepared using thefollowing procedure, the procedure is particularly suitable for melanomaand ovarian carcinoma. A dissected tumor is transferred to a biosafetycabinet and tested for sterility. The tumor is removed from thetransport medium with sterile forceps and submerged successively for 1minute each in three specimen containers with 50 ml of sterile Hankssolution. The tumor is then transferred to a Petri dish for weighing.

Five to 10 ml of Hanks solution is added, and tumors are cut with ascalpel into pieces of approximately 3 mm or less in diameter. Thesolution is removed with a pipette, and the tumor pieces are transferredto a sterile, disposable baffled flask and a sterile magnetic stir baris added. Tumors are digested with 50 ml wash and thaw (500 ml Hanksbuffer+0.5 g EDTA+2 ml 25% HSA, adjusted to pH to 7.2 by addition of 1 NNaOH) containing 70 mg collagenase. Collagenase solutions are filteredthrough 0.2 mm filters prior to use. Digestion is allowed to proceed for30 min at 37° C. with stirring. After digestion, the tumor is filteredthrough a wire mesh screen and the flow through containing the cells iscollected.

Cells are pelleted by centrifuging for 7 minutes at 276×g at roomtemperature and re-suspended in 20 ml Hanks solution without HSA. Cellsare pelleted by centrifuging for 7 minutes at 276×g at room temperatureand re-suspended in 10 ml Hanks solution without HSA. A 10 ml aliquot isremoved aseptically and diluted in 0.2% Trypan blue (25 ml 0.4% Trypanblue stock+25 ml PBS Ca/Mg free). Cells are enumerated in ahemacytometer and the numbers of trypan blue excluding tumor cells (I),trypan blue staining tumor cells (NI) and lymphocytes (L) will berecorded (after digestion and cell count). Optionally, the cells can beirradiated. The cells may also be haptenized, as described below, byadding DNFB stock solution and incubating the cells for 30 min at roomtemperature with mixing every 10 minutes by inversion of the tubes

Cells are thereafter pelleted by centrifuging for 7 minutes at 276× g atroom temperature. All the supernatant (10 mL) is transferred and placedin a sterile, labeled tube at 4° C. This sample is tested for sterility.The cells are re-suspended in 2 ml sterile freezing medium (60 ml Hankssolution+8 g sucrose+40 ml 25% HSA, adjusted to pH 7.2; filter through a0.2 mm filter). In cases where there are a very large number of cells,re-suspend in the minimum volume possible. Preferably, the finalconcentration of cells is about 25×10⁶ cells/mL. The cell vials areplaced into a Nalgene Cryo 1° C. Container containing isopropanol andplaced in the −80° C. freezer overnight. The next day, the vials aretransferred to liquid nitrogen for storage.

Example 2 Distribution of Cell Types After Digestion, Haptenization andFreezing and Thawing

Melanoma vaccines are relatively uniform with respect to cellularcomposition, i.e., proportion of intact cells, non-intact cells andlymphocytes, despite the natural variability of the starting tumors.Freezing and thawing of the vaccine results in significant conversion ofintact cells to non-intact cells. However, irradiation during thepreparation of the vaccine does not affect the cellular composition ofmelanoma vaccines.

The cellular compositions of melanoma vaccines at three stages ofmanufacture, after digestion, after haptenization, and after freezingand thawing, are presented in Table 1. As expected because each tumor isa unique entity, there was considerable variability among the individualtumors at the first point at which a count was done, i.e.,post-digestion. However, for a given vaccine, the proportion of eachcell type changed little as a result of haptenization, but significantchanges were observed following freezing and thawing. TABLE 1 Cell Type(%)¹ I NI L After After After After After After After After AfterVaccine Digest Hapten Thaw Digest Hapten Thaw Digest Hapten ThawNon-Irradiated LTB 12.2 6.1 1.8 41.8 74.0 86.3 46.0 19.9 11.9 ES 20.516.7 2.3 31.8 16.2 90.1 47.7 67.2 7.7 VD 5.1 3.7 8.2 46.1 29.9 77.4 48.866.4 14.4 PAG 50.7 39.2 4.2 14.5 19.1 79.2 34.8 41.7 16.6 RLT 6.7 2.07.7 47.1 12.1 77.6 46.2 85.9 14.7 PSB 2.2 3.8 1.1 79.8 78.7 69.0 18.017.5 29.9 MD 10.3 11.6 1.2 67.3 39.5 88.1 224 48.8 10.7 RM 28.6 38.320.0 46.4 41.3 70.6 25.0 20.3 9.4 EM 12.9¹ 13.6 2.2 70.0¹ 74.6 86.017.1¹ 11.9 11.8 DUI 7.3 7.2 3.2 69.9 71.7 80.5 22.8 21.1 16.3 TB 41.253.1 2.8 27.5 26.5 85.5 31.4 20.4 11.7 TWL NT NT NT NT NT NT NT NT NTMean Std Dev Irradiated JHW 16.2 15.7 1.3 19.1 12.7 79.2 64.7 71.6 19.5JD2 32.0 27.0 4.1 51.5 30.1 82.1 16.6 42.9 13.9 VFE 38.5 43.6 10.9 21.420.1 80.5 40.1 36.2 8.7 CP 10.7 17.3 0 33.1 40.0 77.9 56.2 42.7 22.1 LD16.2 14.8 3.8 54.5 48.1 79.2 29.2 37.0 17.0 Mean 22.7 23.7 4.0 35.9 30.279.8 41.4 46.1 16.2 Std Dev 11.9 12.2 4.2 16.5 14.4 1.6 19.6 14.6 5.2Non-Irradiated Mean 18.8 19.6 4.7 45.0 39.7 80.6 33.9 40.7 14.8 Std Dev14.5 15.9 5.0 19.8 25.0 5.8 15.0 22.3 5.6NT = Not Tested;NA = Not Applicable¹I = Intact Cells (trypan blue excluding), NI = Non-Intact Cells (trypanblue positive), L = Lymphocytes2 Digestion solution included gentamycin3 TCE vaccine passed test; TCM vaccine failed test

Results: The small change in the proportion of each cell type as aresult of haptenization is reflected in the similar resultspost-digestion and post-haptenization, both for individual vaccines andfor the means. The significant changes as a result of freezing and thenthawing of the vaccine are reflected in the observed decreases of intacttumor cells (I) with concomitant increases in the amount of non-intact(NI) tumor cells as well as decreases in the proportion of lymphocytes.This change was observed in all cases except those vaccines for which alow percent of intact tumor was observed initially (patients VD, RLT andPSB).

Overall, the variability in the proportions of different cell typesamong the post-thaw samples of the vaccines was much less than forpost-digestion and post-haptenization samples, as reflected in the muchsmaller standard deviation, demonstrating that the vaccines wererelatively uniform despite the variability of the starting tumors.Further, there was no obvious difference between those vaccines thatwere irradiated during preparation versus those that were not. The meanproportions of the different cell types in the various fractions wasvery similar for irradiated samples alone compared to the meanproportions for all samples, irradiated or not.

Example 3 Retention of HLA Class I Antigen and Surface DNP AfterSucrose-Freezing Medium Treatment and Cryopreservation

This Example describes the cell recovery and antigenicity of haptenizedcells when treated with freezing medium, comprised of sucrose, humanserum albumin and Hank's buffered solution and stored in liquid nitrogenfor up to 7 months. Both irradiated and non-irradiated vaccine sampleswere tested. Cell counting and flow cytometry were conducted asdescribed below.

As shown in the table below, sucrose freezing medium treatment followedby cryopreservation resulted in little evidence of a significantdiminution of surface DNP or surface HLA class I antigen. TABLE 2Stability of Surface DNP and HLA Class I of Melanoma Vaccine Stored inLiquid Nitrogen for up to 7 Months Shift (%) Surface DNP HLA Class ITotal Back- Total Shift Back- HLA Time Shift ground DNP- (HLA groundClass I- Vaccine Point (mo) (Anti-DNP) (Isotype) Isotype Class I)(Isotype) Isotype Non-Irradiated LTB 0 82.35 24.00 58.35 71.99 24.0047.99 3.5 91.45 24.15 67.30 73.67 24.15 49.52 7 86.15 25.14 61.01 66.0125.14 40.87 ES 0 63.48 23.24 40.24 98.02 19.48 78.54 VD 0.5 85.65 26.9058.75 98.60 26.90 71.70 3 36.36 19.89 16.47 96.20 19.89 76.31 5.2 40.2924.10 16.19 94.14 24.10 70.04 PAG 3 88.58 14.78 73.80 89.18 14.78 74.405.2 87.69 12.12 75.57 88.39 12.12 76.27 RLT 0 89.80 14.75 75.05 90.7014.75 75.95 3 86.50 29.14 57.36 86.77 29.14 57.63 6 86.84 13.46 73.494.89 13.46 81.43 PSB 0 97.62 38.47 59.15 96.27 38.47 57.80 3 85.3328.05 57.28 88.90 28.05 60.85 MD 0 96.62 35.54 61.08 98.26 35.54 62.72 492.84 23.64 69.20 91.49 23.64 67.85 RM 0 66.32 27.50 38.82 97.28 27.5069.78 DUI 0 80.82 21.53 59.29 72.29 21.53 50.76 2 64.37 24.14 40.2363.37 24.14 56.99 3 85.42 27.71 58.25 79.67 27.71 51.96 TB 0 82.73 12.3070.43 83.71 12.30 71.41 1 86.22 24.67 61.55 91.24 24.67 66.57 3.5 94.5526.48 68.07 92.56 26.48 66.08 Irradiated JD2 0 79.03 14.53 64.50 52.3014.53 37.77 1 92.63 30.82 61.81 65.28 30.82 34.46 3.5 97.84 20.09 77.7566.04 20.09 45.95 VFE 0 67.72 11.79 55.93 98.09 11.79 86.30 2 89.6019.03 70.57 98.94 19.03 79.91 3 89.84 18.51 71.33 98.90 18.51 80.39 CP 051.53 27.37 24.16 98.94 27.37 71.57 1 81.34 18.10 63.33 96.47 18.1078.37 2.5 26.09 28.35 0.00 99.48 28.35 71.13 LD 0 76.95 26.01 50.9485.04 26.01 59.03 2 78.75 24.24 54.51 77.70 24.24 53.46 3 79.67 26.0553.62 79.93 26.05 53.88 Irradiated LC 0 87.06 18.86 68.20 54.46 18.8635.60 2 95.54 20.23 75.31 53.55 20.23 33.32 JHW ¹ 0 44.91 22.45 22.4697.50 22.45 75.05 1.5 59.86 48.79 11.07 98.90 48.79 50.11 2 44.61 21.2223.39 98.16 21.22 76.94 4 48.64 21.49 27.15 97.99 21.49 76.50¹ Due to calculation error, received only half the amount of DNFB

Protocol: The irradiated and non-irradiated haptenized cells werecontacted with freezing medium. The freezing medium was comprised of 8%sucrose, 10% human serum albumin and Hank's buffered solution. Thesamples were frozen overnight in a −80° C. freezer and then transferredto a liquid nitrogen freezer for storage.

Results: The results for both the irradiated and non-irradiatedhaptenized tumor cells shows that the surface hapten DNP or surface HLAclass I antigen was maintained over several months of storage. Only onesample, designated VD in Table 2, showed any consistent loss ofhaptenization and freezing medium treatment and long-term storage inliquid nitrogen.

Cytometry Procedures

The tumors samples were removed from liquid nitrogen, placed at −196° C.overnight and then thawed in a 37° C. water bath for approximately 90sec and placed on ice. The samples may be filtered at this time using a70 μm Falcon nylon filter. The samples were split into 7 tubes, 3 tubesfor surface staining and 4 tubes for internal staining. The surfacesamples were washed in 1 ml/tube of wash buffer (phosphate-bufferedsaline with 0.1% bovine serum albumin and 0.1% NaN3) and cells werepelleted at 1500 rpm for 7 minutes at 4° C. All but 100 μl of the washwas discarded and 1.1 μg of either mouse anti-human HLA-ABC (Dako cat#M0736; IgG2a); mouse anti-DNP (Sigma cat #8406), or a mouse IgG2acontrol isotype control antibody was added to the samples. Afterincubation for 1 hr at 4° C., the cells were washed in 3 ml of washbuffer and pelleted at 1500 rpm for 7 minutes at 4° C. All but 100 μl ofthe wash was aspirated and 5 μl of secondary antibody, rabbit anti-mouseIg conjugated with fluorescein isothiocyanate (FITC, Dako cat #F0313)was added to each sample tube. After incubation for 45-60 min at 4° C.,the samples were washed one more time with 3 ml of wash buffer and cellswere pelleted at 1500 rpm for 7 minutes at 4° C. After the last wash,all but 100 μl of the wash was aspirated from the tubes and cells werere-suspended in 2 ml of wash buffer. Samples were read on a BeckmanCoulter Epics Altra flow cytometer and the data was analyzed using Expo32® software (Applied Cytometry Systems). The shift was determined bycalculating the percentage of events that had a fluorescence greaterthan that at the half-maximum peak height on the right-hand side of thecurve for the isotype control antibody, as described by Erdile et al.,(J. Immunol. Meth., 2001; 258:47-53).

Example 4 Stability of Sucrose Freezing Medium-Treated CryopreservedCells

This Example compares long-term stability of irradiated andnon-irradiated DNP-haptenized melanoma cells. Briefly, cells weresuspended in freezing medium comprising 8% sucrose, 10% human serumalbumin and Hank's buffered solution, placed in a −80° C. freezerovernight, and transferred to a liquid nitrogen freezer for storage. Atvarious time points up to 9 months, samples were thawed and the relativefractions of intact cells, non-intact cells, and lymphocytes weredetermined. The results are shown in Table 3.

The percentages of the different cell types remained relativelyunchanged over at least the first 3 months of storage, and the variationbetween irradiated and non-irradiated tumor cell samples was small. Theresults show that the freezing medium preserves haptenized tumor cells.TABLE 3 Stability of Cellular Composition of Melanoma Vaccine Stored inLiquid Nitrogen Up to 9 Months Time Cell Type (%)¹ Vaccine (Months) I NIL Non-Irradiated LTB 0 1.8 86.3 11.9 0.5 2.6 80.9 16.5 1 2.5 83.0 14.5 22.3 84.7 13.0 3 3.9 94.4 1.7 7 2.9 89.7 7.4 9 1.9 93.3 4.8 VD 0 8.2 77.414.4 0.5 8.0 59.4 32.6 1 4.2 79.0 16.8 2 3.2 84.2 12.6 3 0.5 81.4 18.05.5 0.8 78.4 20.8 PAG 0 4.2 79.2 16.6 0.5 4.0 87.0 9.1 1 5.2 45.0 49.8 27.5 86.3 6.2 3 2.9 78.6 18.4 RLT 0 7.7 77.6 14.7 0.5 6.0 88.0 6.0 1 12.083.7 4.2 2 5.5 75.2 19.3 3 5.0 69.5 25.5 5.5 6.5 79.7 13.8 PSB 0 1.169.0 29.9 0.5 3.7 46.3 50.0 1 0.5 87.5 12.0 2 1.8 79.0 19.2 3 3.0 74.722.3 6 0/0 87.5 12.5 MD 0 1.2 88.1 10.7 0.5 0.5 49.5 50.0 1 0.0 88.711.3 2 1.4 61.4 37.2 3 0.0 66.7 33.3 4 1.2 85.5 13.3 5.5 0.0 93.1 6.9DU1 0 3.2 80.5 16.3 0.5 0.0 87.4 12.6 1 0.0 91.7 8.3 2 2.3 91.9 5.8 34.9 76.9 18.2 TB 0 2.8 85.5 11.7 0.5 5.1 78.1 17.7 1 1.9 75.8 22.8 2 2.082.0 16.4 3.5 0.7 88.1 11.2 Irradiated JHW 0 1.3 79.3 19.5 1 5.2 86.78.6 1.5 0.0 80.2 19.8 2 5.1 68.5 27.8 4 1.1 93.0 5.9 JD2 0 4.1 82.1 13.90.5 2.2 86.7 11.4 1 0.0 85.0 15.0 2 5.3 75.8 19.9 3.5 5.4 74.5 21.3 VFE0 10.9 80.5 8.7 1 6.2 69.2 26.2 2 3.0 86.5 10.8 3 4.1 84.0 12.4 CP 0 0.077.9 22.1 0.5 0.8 76.8 22.6 1 0.0 83.2 16.8 2 0.0 90.8 9.2 2.5 0.0 92.27.8 LD 0 3.8 79.2 17.0 1 10.0 75.0 16.7 2 10.4 85.7 4.3 3 0.0 87.1 12.9LC 0 2.9 81.2 15.9 1 4.9 81.7 14.1 2 1.8 93.0 5.3¹I = Intact Cells (trypan blue excluding), NI = Non-Intact Cells (trypanblue positive), L = Lymphocytes

Example 5 In Vivo Efficacy of Cryopreserved Vaccine

This Example describes the evaluation of the therapeutic efficacy offrozen dinitrophenyl (DNP)-modified tumor cell vaccine against tumorrecurrence in mice from which a primary 410.4 mammary carcinoma wassurgically excised. The results show that frozen DNP-modified tumor cellvaccine is equally effective in improving relapse-free survival as afresh DNP-modified tumor cell vaccine (Sojka et al., Cancer Immunol.Immunother. 2002; 51:200-208). The improvement in relapse-free survivalwas consistent between 4 independent experiments, and, when the datafrom all 4 studies were pooled, the effect was statisticallysignificant. A direct comparison of frozen and fresh DNP-modified tumorcell vaccine suggested their efficacy was indistinguishable. Further,the relapse-free survival of mice immunized with frozen DNP-modifiedtumor cell vaccine was also superior to that of mice immunized withfrozen unmodified tumor cell vaccine in 3 independent experiments, andthe difference was statistically significant when the data from all 3studies were pooled. Accordingly, DNP-modification improves thetherapeutic benefits of this tumor cell vaccine, and thecryopreservation method employed does not diminish the vaccine potency.Preliminary results also indicated that subjecting the tumor cells togamma irradiation prior to their DNP modification improved vaccineefficacy. The vaccine was well-tolerated, showing only mild,self-limited induration at the injection site. The materials andexperimental design, as well as the results, are discussed in moredetail below.

The animal model employed the highly metastatic 410.4 tumor thatoriginates from a spontaneously arising BALB/c murine mammary carcinoma.Tumor cells were maintained in vitro as previously described (Sojka etal., Cancer Immunol. Immunother. 2002; 51:200-208), and 3×10⁵ tumorcells were injected into mammary fatpads of the left breast of femaleBALB/cAnNCrBR mice 7-10 weeks old (Charles River Breeding Laboratories,Wilmington, Mass.). Tumors were allowed to grow to 6-8 mm in diameter,at which point the primary tumor was surgically excised and treatmentwas initiated. The mice were monitored at least twice a week for localtumor recurrence and for the appearance of palpable metastases in theother breast and in the regional lymph nodes.

For vaccine preparation, tumor cells were detached from the cultureflasks with EDTA (Sigma Chemical Co., St Louis, Mo.), and subjected toγ-irradiation (2500 cGy from a ¹³⁷Cs source), except where indicated.Haptenization was performed by the addition of 2,4-dinitrofluorobenzene(DNFB, Sigma Chemicals Co., St. Louis, Mo.) with incubation for 30 minat room temperature to prepare clinical vaccines from human tumors. Forfresh DNP-modified, γ-irradiated, tumor cell vaccine the cells were usedon the day of the preparation. For the frozen DNP-modified,γ-irradiated, tumor cell vaccine, the DNP-modified, γ-irradiated, tumorcells were washed and re-suspended in Hanks' solution supplemented with8% sucrose and 10% human serum albumin (HSA). The DNP-modified cellswere then aliquoted, and the vials were placed into a Nalgene Cryo 1° C.Containers containing isopropanol and placed into a −80° C. freezerovernight. The frozen cells were then stored until use. Immediatelyprior to use, the cells were thawed in a 37° C. water bath and washedtwice in Hanks' solution to remove residual HSA. Frozen unmodified,γ-irradiated, tumor cell vaccine was prepared in the same fashion,except that no DNP modification was performed. Each vaccine wasadministered in a total volume of 0.2 ml and consisted of 5×10⁶ frozenDNP-modified tumor cells or unmodified tumor cells, or 3×10⁶ freshDNP-modified tumor cells admixed with 0.5×10⁶ to 4×10⁶ colony-formingunits (CFU) of Bacille Calmette-Guerin (BCG, Tice strain, Organon).

According to the experimental design, three to five days after surgicalexcision of the primary tumor, the mice were given an i.p. injection of15 mg/kg cyclophosphamide (CY; Mead Johnson—A Bristol-Myers Squibb Co.,Princeton, N.J.). Three days after the low-dose CY treatment, the micereceived a s.c. injection of the indicated vaccine close to the site oftumor excision. This protocol was repeated every 10 days for theduration of the experiment. As a reference point, in some experiments agroup of mice received saline. All mice were monitored at least twice aweek for tumor recurrence at the primary site as well as for theappearance of palpable metastases in the other breast and in theregional lymph nodes. Once metastases were evident, the progression ofmetastases was followed for the duration of the experiments, or untilthe mice showed signs of distress, after which they were sacrificed. Inaddition to monitoring for tumor recurrences, in some experiments, theanimals were also monitored for injection site reactions and weight.

Statistical analysis of the data was conducted was conducted by plottingthe fraction of animals free of tumor recurrence at each monitoringpoint as Kaplan-Meier type survival curves, and a log rank test wasperformed using GraphPad software from Prism software, San Diego, Calif.A p value of 0.05 or less was considered significant.

The results showed that frozen DNP-modified tumor cell vaccine improvedrelapse-free survival as compared to saline injection. A total of fourexperiments were carried out, each demonstrating the therapeuticbenefits of the frozen DNP-modified tumor cell vaccine relative to thesaline treatment group, and the results pooled. The pooled results,showing a statistically significant difference between the groups(p=0.0011) are shown in FIG. 1. Thus, treatment of mice from which theprimary tumor was surgically excised with frozen DNP-modified tumor cellvaccine offers therapeutic benefits against tumor recurrence.

In independent experiments, fresh and frozen DNP-modified tumor cellvaccines were compared for their therapeutic effectiveness against tumorrecurrence in 410.4 tumor. When the results of the experiments werepooled, the two treatment groups offered comparable therapeutic benefits(p=0.6725). These results are displayed in FIG. 2. The therapeuticeffectiveness of the frozen DNP-modified tumor cell vaccine alsoappeared to be similar to the therapeutic effectiveness of the freshDNP-modified tumor cell vaccine reported by Sojka et al., (CancerImmunol. Immunother. 2002; 51:200-208).

In addition to comparing the relapse-free survival of mice receivingfrozen DNP-modified tumor cell vaccine to that of the saline treatmentgroup, experiments were carried out to compare the relapse-free survivalof mice receiving frozen DNP-modified irradiated tumor cell vaccine tothat of mice receiving frozen unmodified irradiated tumor cell vaccine.When all studies were pooled, the DNP-modified frozen vaccine inducedsignificantly better relapse-free survival (p=0.0004). These results areshown in FIG. 3. Taken together, our current results show that frozenDNP-modified irradiated tumor cell vaccine, like fresh DNP-modifiedirradiated tumor cell vaccine, is superior to unmodified irradiatedtumor cell vaccine in improving the relapse-free survival of mice fromwhich the primary 410.4 tumor was surgically excised, implying that DNPmodification is essential to producing optimal anti-tumor effects.

Further, experimental results indicated that irradiation was importantto the effect of DNP-modified tumor cell vaccine. Haptenization withDNP, with or without irradiation, has been shown to be sufficient toeliminate both in vitro proliferation and in vivo tumorigenicity. Toexamine whether this might allow for elimination of the irradiationstep, frozen DNP-modified tumor cell vaccines prepared with or withoutirradiation were compared. The results indicated a significantly lowerrelapse-free survival for non-irradiated than frozen DNP-modifiedirradiated tumor cell vaccine (FIG. 4; p=0.0362). The mechanism(s)through which irradiation is improving the efficacy of DNP-modifiedtumor cell vaccine against tumor recurrence in the 410.4 tumor systemremains to be elucidated. Irradiation has, however, been shown toactivate the expression of genes (e.g., B7-1 (Morel et al., CancerImmunol Immunother 1998; 46:277-282; Sojka et al., J Immunol 2000;164:6230-6236; Vereecque et al., Br J. Haematol 2000; 108:825-831) andTNF-α (Hallahan et al., Proc. Natl. Acad. Sci. (USA) 1989;86:10104-10107; Weill et al. J Interferon Cytokine Res 1996;16:395-402)) that are known to be important for the acquisition of tumoreradicating immunity (Chen et al., Immunol Today 1993; 14:483-486;Allison et al., Curr Opin Immunol 1995; 7:682-686; Gorelik et al., JImmunol 1995; 154:3941-3951; Sojka et al., Cancer Immunol. Immunother.2002; 51:200-208), which might explain these results.

Finally, the DNP-modified tumor cells vaccines, whether fresh or frozen,were well tolerated. Animals that had received frozen DNP-modifiedirradiated tumor cell vaccine maintained their weight in a similarfashion to control animals that received saline. The onlyvaccine-related adverse reactions noted in these studies were edema andinduration at the injection site, most likely associated with the BCGcomponent of the vaccine. These were mild and self-limited anddisappeared before the subsequent vaccination.

Example 6 Cryopreserved Bi-Haptenized Cells

This Example describes the cell recovery, antigenicity, and haptenretention of bi-haptenized melanoma cells when treated with sucrosefreezing medium, comprised of sucrose, human serum albumin and Hank'sbuffered solution and stored in liquid nitrogen for up to 6 months.

Bi-haptenized melanoma cell compositions were prepared by conjugatingapproximately half of a tumor cell preparation with DNP, and the otherhalf with SA, as described above. The sucrose freezing medium wasprepared by mixing 60 ml HBSS with 40 ml 25% HSA and 8 g sucrose untilthe sucrose was completely dissolved, followed by sterile-filtrationinto a disposable plastic bottle using a 0.2 μm filter apparatus.Stability testing by cell counting and flow cytometry was essentiallyconducted as described in Example 3, testing for expression of the cellmarkers HLA Class I, CD45, GD3, S100, HMB45, and Mart-1. Tables 5 and 6below show the results of the evaluation for 12 samples, denoted #1-12,each representing a different tumor cell preparation. In essence, therewere no consistent changes in the cryopreserved cell preparations for upto 6 months of storage. TABLE 5 Stability of Mixed Haptenized Vaccine -Cell Counts # Large Time Cell Counts* # Cells/Vial Frozen Large Small 12.5 1 D 3.4 4.6 2 W 3.6 7.3 2 M 2.4 5.6 4 M 2.1 4.5 6 M 1.5 5.4 2 2.5 1D 3.7 0.7 2 W 3.3 0.3 2 M 1.6 0.4 4 M 1.7 0.4 6 M 1.2 0.4 3 5 1 D 2.50.4 2 W 2.3 0.2 2 M 2.1 0.4 4 M 2.6 0.4 6 M 2.2 0.2 4 2.5 1 D 2.5 0 2 W1.1 0.1 1 M 2.4 0.1 2 M 2.6 0.2 3 M 2.2 0 5 1.25 1 D 0.9 0.1 2 W 1.7 0 1M 1.1 0.1 2 M 1.3 0.2 3 M 2.1 0.3 6 1.25 1 D 1 0 2 W 0.9 0.1 1 M 1.1 0 2M 1.3 0.1 3 M 0.7 0 7 2.5 1 D 1.9 3.2 2 W 2.4 3.4 1 M 2.8 2.6 2 M 2.61.8 3 M 2.3 3.6 6 M 1.3 2 8 2.5 1 D 2.8 9.3 2 W 3.1 9.4 1 M 2.5 6 2 M 28.7 3 M 2.3 6.1 6 M 2.9 7.2 9 1.25 1 D 2.5 23 2 W 1.6 11.5 1 M 1.2 17.32 M 1.8 18.3 3 M 1.7 11.7 6 M 1.7 11 10 1.25 1 D 1.3 7.6 2 W 1 6.9 1 M1.1 5.2 2 M 1.5 9.7 3 M 1.4 5.5 6 M 0.6 5.1 11 1.25 1 D 1.9 0.6 2 W 10.8 1 M 0.8 1.1 2 M 1 1.1 3 M 1.4 0.7 6 M 1.2 0.8 12 1.25 1 D 1.6 6.5 2W 1.1 5.8 6 W 1 4.3 8 W 1.4 6.2 3 M 1.5 6.4 6 M 0.5 4.7*all cell counts expressed in millionsD = Day(s); W = Week(s); M = Month(s)

TABLE 6A Stability of Mixed Haptenized Vaccine - Retention of HLA ClassI, CD45, GD3, and S100 Antigens Time HLA Class I CD45 GD3 S100 # Frozen% (+)# Peak@ % (+) Peak % (+) Peak % (+) Peak 1 1 D 54.4 5.6 41.1 4.454.5 5.2 25.8 5.8 4 M 44.1 2.3 15.1 2.5 26.8 2.5 21.2 2.5 6 M 69.0 2.042.8 1.9 38.9 2.0 42.3 1.9 2 1 D 10.8 3.8 9.5 4.2 66.9 8.0 58.7 4.4 4 M13.4 1.5 6.3 1.5 46.2 1.5 58.0 2.2 6 M 12.9 2.2 7.8 2.3 68.7 4.2 62.92.2 3 1 D 14.0 4 M 15.2 2.1 8.8 1.8 93.9 20.1 69.0 2.2 6 M 13.1 2.3 2.82.1 89.7 11.1 53.9 2.1 4 1 D 62.1 4.3 5.8 3.7 98.3 34.2 93.0 8.1 1 M40.9 2.9 5.8 3.0 96.9 16.1 89.2 9.4 3 M 56.8 2.5 5.5 1.8 97.9 17.3 89.94.2 5 1 D 30.2 4.9 2.3 5.9 86.1 38.4 73.7 5.1 1 M 33.8 2.5 3.4 2.2 96.132.1 83.7 4.2 3 M 34.9 2.8 2.8 1.9 90.6 22.0 88.1 5.2 6 1 D 69.6 1.6 9.90.7 93.8 21.8 97.0 31.8 1 M 58.9 1.8 4.3 0.6 92.5 16.1 93.7 24.3 3 M59.3 2.8 4.7 1.2 97.7 41.7 85.7 7.9 7 1 D 43.3 219.0 26.9 1.4 42.0 8.654.2 4.9 2 W 35.2 3.8 17.5 3.2 80.3 17.4 52.8 3.6 1 M 25.4 2.3 11.2 2.223.1 7.1 43.6 3.0 2 M 40.4 2.4 21.1 2.5 60.3 2.7 84.5 5.2 3 M 76.1 0.934.4 3.7 90.3 10.5 98.7 4.4 6 M 38.7 1.9 48.2 1.7 44.1 5.6 53.3 2.8 8 1D 24.8 4.0 11.9 3.8 38.1 4.6 39.2 4.3 2 W 18.0 6.4 14.6 6.7 57.3 7.248.3 6.8 1 M 26.6 2.3 16.8 2.2 51.7 2.5 60.7 2.2 2 M 28.8 1.9 18.3 1.948.8 1.9 58.6 2.1 3 M 56.2 1.6 58.7 2.4 78.1 1.8 87.7 3.2 6 M 21.4 4.814.2 4.9 35.4 5.1 45.1 6.5 9 1 D 87.0 4.1 89.5 1.6 71.4 1.3 48.5 1.0 1 M64.4 6.7 14.8 6.0 54.1 38.4 41.9 17.1 2 M 76.6 6.4 45.8 3.8 79.1 6.233.2 3.9 3 M 95.3 5.1 68.8 3.4 59.8 1.7 95.2 3.8 6 M 55.1 3.2 29.7 2.538.1 2.5 28.0 2.7 10 1 D 65.9 4.9 30.5 3.6 14.3 3.6 18.2 3.5 2 W 66.25.2 53.3 2.3 47.4 4.1 22.1 2.2 1 M 80.9 2.6 68.2 1.6 40.1 1.2 31.5 1.3 2M 78.1 3.0 51.1 2.3 33.6 1.6 22.7 1.4 3 M 77.5 7.0 60.5 3.7 19.7 6.730.4 4.7 6 M 81.9 5.3 43.5 2.1 33.0 2.2 35.8 2.1 11 1 D 52.1 3.4 26.31.9 79.0 10.6 77.5 5.8 2 M 39.8 1.9 17.8 2.1 98.4 19.6 96.0 10.3 3 M55.2 0.9 20.7 3.4 95.3 3.1 98.7 6.8 6 M 38.8 1.7 37.6 1.2 86.3 8.6 83.34.8 12 1 D 67.1 2.7 55.2 2.6 79.6 4.6 68.7 3.7 6 W 62.1 2.5 36.6 2.239.7 1.8 41.6 2.3 8 W 66.6 3.0 35.1 2.2 51.2 1.8 57.7 2.6 3 M 81.9 2.233.3 1.2 45.6 1.0 81.4 3.6 6 M 77.3 4.6 57.8 2.6 49.7 3.2 62.3 3.5#% (+) indicates the percentage of cells expressing the marker@Peak indicates peak fluorescence channel of positive cellsD = Day(s);W = Week(s);M = Month(s)

TABLE 6B Stability of Mixed Haptenized Vaccine - Retention of HMB45,MART-1 Antigens, and DNP and SA Haptens Time HMB45 MART-1 DNP SA #Frozen % (+) Peak % (+) Peak % (+) Peak % (+) Peak 1 1 D 5.4 4.6 11.033.6 53.9 16.2 55.3 4.1 4 M 1.5 2.9 12.7 3.2 52.1 7.1 22.2 3.2 6 M 2.32.5 10.3 2.2 56.6 5.6 33.4 2.3 2 1 D 6.4 3.2 12.7 3.4 60.6 35.1 34.4 3.54 M 6.0 1.8 9.9 1.5 56.6 6.4 30.0 7.3 6 M 8.4 2.1 14.3 3.0 63.1 12.527.7 12.0 3 4 M 13.3 1.8 14.2 1.8 71.0 18.0 34.1 1.9 6 M 11.3 2.0 4.42.0 82.6 18.2 21.8 12.9 4 1 D 2.2 3.8 3.0 4.0 63.1 35.4 41.2 35.7 1 M2.6 3.4 1.7 2.9 62.4 34.5 34.5 8.1 3 M 3.8 2.0 3.7 2.0 65.7 32.1 37.18.6 5 1 D 8.0 5.3 30.9 5.5 63.1 37.7 41.8 11.8 1 M 8.3 3.4 38.1 3.0 59.126.6 40.1 4.7 3 M 6.6 1.7 43.1 2.8 54.0 11.6 42.1 6.9 6 1 D 84.8 1.054.9 1.7 48.7 8.2 41.5 32.1 1 M 71.1 1.4 42.8 0.9 44.8 4.2 44.7 30.1 3 MNA NA 44.2 2.5 43.7 5.8 40.2 20.5 7 1 D 1.5 1.6 2.2 1.4 47.0 8.7 27.13.1 2 W 3.2 3.2 1.6 3.2 51.4 29.3 54.7 8.7 1 M 1.8 2.3 6.2 2.5 54.2 7.932.8 4.0 2 M 2.4 2.1 1.7 2.1 54.3 20.5 55.5 5.0 3 M 7.3 0.7 7.4 0.8 54.514.9 59.1 3.8 6 M 2.3 1.9 1.5 2.3 58.3 11.0 35.7 4.5 8 1 D 1.4 3.4 7.53.4 43.8 7.0 23.2 3.8 2 W 2.9 7.0 17.0 6.8 52.3 12.6 25.4 8.0 1 M 2.92.4 10.5 2.3 54.1 6.4 NA NA 2 M ND ND 9.7 2.0 52.0 10.0 35.4 2.5 3 M 4.31.4 23.3 4.1 55.3 12.1 46.4 7.7 6 M 3.9 5.2 9.3 5.0 45.2 6.7 23.8 6.2 91 D 3.6 0.9 2.4 1.1 42.5 9.1 51.8 2.7 1 M 0.7 5.9 16.9 6.3 53.3 28.057.1 18.9 2 M 1.9 4.0 17.8 6.1 54.2 12.0 62.5 7.0 3 M 20.3 6.4 57.1 18.266.2 4.2 6 M 1.3 2.3 11.2 4.6 51.4 8.4 61.5 1.5 10 1 D 0.9 3.8 1.7 3.561.5 14.7 39.9 10.4 2 W 1.6 2.5 4.2 2.3 58.9 10.6 83.4 6.5 1 M 3.5 1.33.6 1.2 60.9 7.0 39.2 1.9 2 M 1.9 1.4 2.8 1.4 60.9 5.8 42.6 2.4 3 M 1.64.5 4.4 4.3 70.6 13.8 41.9 6.6 6 M 3.2 2.4 2.2 2.7 69.7 7.6 28.3 2.6 111 D 23.9 2.0 28.6 2.4 45.7 23.4 36.0 20.1 2 M 21.9 1.8 23.7 2.0 52.621.4 44.5 34.5 3 M NA NA 42.0 1.9 52.5 30.4 67.3 33.3 6 M 13.4 1.7 14.61.6 48.9 13.4 42.0 25.2 12 1 D 2.1 2.2 9.3 2.5 56.8 13.1 44.7 4.2 6 W3.7 1.7 23.1 16.3 62.6 6.7 36.4 1.7 8 W 5.1 1.9 18.7 25.2 67.0 5.5 41.31.9 3 M NA NA 28.2 5.9 63.7 5.1 26.7 2.1 6 M 3.1 2.3 24.9 10.0 68.3 8.533.2 2.3# % (+) indicates the percentage of cells expressing the marker@Peak indicates peak fluorescence channel of positive cellsD = Day(s);W = Week(s);M = Month(s)

Example 7 Clinical Studies with Cryopreserved Cells

This Example outlines various clinical studies using freezing mediumtreated cryopreserved haptenized cells.

A novel human cancer vaccine, consisting of autologous tumor cellsmodified with the hapten, dinitrophenyl (DNP), has been developed. TheDNP-modified vaccine induces unique immunological effects and showsclinical efficacy. A second-generation vaccine composed of autologoustumor cells, half of which have been modified with DNP and half with asecond hapten, sulfanilic acid (SA), has also been developed. This“bihaptenized” vaccine is immunologically more potent and clinicallymore effective.

A phase I trial of the bihaptenized vaccine in patients with stage IVmelanoma is conducted, testing four dosage levels. The major endpointsare the development of delayed-type hypersensitivity (DTH) toDNP-modified, SA-modified, and unmodified autologous tumor cells. Also,the development of tumor inflammatory responses is studied.

Subsequently, a phase II trial using the lowest dose that is found to beimmunologically effective in the phase I trial is conducted. Theimmunological basis of a newly discovered phenomenon—the importance ofthe timing of a vaccine “induction” dose, is investigated. Thehypothesis that the administration of an induction dose timed optimallywith administration of low dose cyclophosphamide results in selectivedepletion of suppressor T cells that would otherwise down-regulate orabrogate the anti-tumor immune response is tested. Peripheral bloodlymphocytes are obtained from patients at various time points andassayed for the presence of suppressor cells. It is then determinedwhether such suppressor cells have a characteristic phenotype, CD4⁺CD25⁺ with co-expression of CTLA4, and whether upon stimulation theyproduce the immunoregulatory cytokine, IL10. Finally, the ability of thesuppressor cells to down-regulate in vitro T cell responses toalloantigens, hapten-modified tumor cells, and unmodified tumor cells,is tested. These studies provide insights into the immunobiology ofhuman cancer vaccines and assist in the development of more effectiveimmunotherapy strategies.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are to some degreeapproximate, and are provided for purposes of description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties.

1. A method of preserving haptenized tumor cells, which methodcomprises: (i) contacting the haptenized tumor cells with a freezingmedium, wherein the freezing medium comprises sucrose, human serumalbumin and an isotonic buffered solution; and (ii) freezing the tumorcells, whereby the immunogenicity of the tumor cells is preserved. 2.The method of claim 1, wherein the isotonic buffered saline solution isHank's buffered solution.
 3. The method of claim 1, wherein the freezingmedium comprises 8% sucrose, 10% human serum albumin and the isotonicbuffered saline solution is Hank's buffered solution.
 4. The method ofclaim 1, wherein the temperature is from about −80° C. to about −196° C.5. The method of claim 1, wherein at least 70% of the level of at leastone tumor cell-associated antigen (TCAA) is preserved after about 3months storage at a temperature of at least −80° C.
 6. The method ofclaim 5, wherein at least 90% of the TCAA is preserved.
 7. The method ofclaim 1, wherein at least 50% of the haptenized tumor cells arepreserved intact after about 3 months storage at a temperature of atleast −80° C.
 8. The method of claim 7, wherein at least 70% of thehaptenized tumor cells are preserved intact.
 9. The method of claim 1,wherein the tumor cells are selected from the group consisting melanomacells, ovarian cancer cells, colorectal cancer cells, small cell lungcancer cells, kidney cancer cells, breast cancer cells, and leukemiacells.
 10. The method of claim 9, wherein the tumor cells are melanomacells.
 11. The method of claim 1, wherein the tumor cells are haptenizedwith at least one hapten selected from the group consisting of DNP, TNP,and sulfanilic acid.
 12. The method of claim 9, wherein the tumor cellsare haptenized with DNP.
 13. The method of claim 11, wherein the tumorcells are haptenized with at least two different haptens.
 14. A methodof storage for haptenized tumor cells for use in a vaccine, which methodcomprises storing a haptenized tumor cells and a freezing mediumcomposition at a temperature below the freezing temperature for at least3 months.
 15. The method of claim 14, wherein the temperature is fromabout −80° C. to −196° C.
 16. The method of claim 12, wherein the tumorcells are haptenized with at least one hapten selected from DNA andsulfanilic acid.
 17. A composition comprising haptenized tumor cells foruse in a vaccine and a freezing medium, wherein the freezing mediumcomprises sucrose, human serum albumin and an isotonic buffered salinesolution.
 18. The composition of claim 15, wherein the freezing mediumcomprises 8% sucrose, 10% human serum albumin and the isotonic bufferedsaline solution is Hank's buffered solution.
 19. The composition ofclaim 15, wherein the tumor cells are selected from the group consistingmelanoma cells, ovarian cancer cells, colorectal cancer cells, smallcell lung cancer cells, kidney cancer cells, breast cancer cells, andleukemia cells.
 20. The composition of claim 17, wherein the tumor cellsare melanoma cells.
 21. The composition of claim 15, wherein the tumorcells are haptenized with at least one hapten selected from the groupconsisting of DNP, TNP, and sulfanilic acid.